Sphingosine-1-phosphate (S1P) is now recognized as a potent lipid mediator that regulates many vital biological processes, including cell growth, death, and differentiation. In a continuing and highly successful collaboration with Spiegel?s lab at Virginia Commonwealth University, we are elucidating the mechanisms by which S1P is produced, how its levels are regulated, and how it mediates its diverse actions. S1P is produced by two sphingosine kinase (SphK) isoforms after activation by diverse types of external stimuli. For example, we found that activation of mast cells by crosslinking of the high affinity IgE receptor not only increased cellular levels of S1P by activating SphK1, an enzyme originally cloned by us, degranulation and motility of RBL 2H3 and bone marrow derived mast cells were dependent on formation and release of S1P which subsequently transactivated specific mast cell S1P receptors. Our results suggest that this process plays a crucial role in mast cell functions in inflammatory responses. In another study with important implications for immunology, we discovered that SphK2 is the enzyme that phosphorylates the novel immunosuppressive agent, FTY720, converting it to a S1P mimetic that is highly effective in preventing organ transplant rejection. Phosphorylated FTY720 acting through S1P receptors prevents egress of activated lymphocytes from secondary lymphoid tissues. Unexpectedly, we found that the second sphingosine kinase isozyme that we cloned, SphK2, has completely the opposite effects on cells as SphK1 has. SphK2 is larger than SphK1, has a different tissue distribution, and contains a putative BH3-only motif, similar to that in a large sub-family of Bcl-2 proteins important for apoptosis. While SphK1 promotes cell growth and survival, SphK2 inhibits cell growth and induces cell death. How these two very closely related and similar enzymes that use the same substrate and produce the same product can have such opposite effects and their importance in regulating cell functions is now under investigation. While many important biological effects of S1P are now known to be mediated by activation of S1P receptors, we have previously suggested that S1P might also have direct intracellular actions. This concept has been challenged due to the scarcity of identified intracellular targets. We have now found that in contrast to treatment of cells with exogenous S1P, which increases proliferation by binding to S1P receptors on the cell surface, increasing intracellular S1P formation stimulated cell growth and promoted survival independently of these receptors. Thus, exogenous and intracellularly generated S1P can affect cell growth and survival by divergent pathways and our study describes a receptor-independent, intracellular function of S1P, reminiscent of its action in yeast and plants that lack S1P receptors. In a study that may have importance for development of the nervous system, we found that differential transactivation of S1P receptors on neurons regulates NGF-induced neurite extension. The process of neurite extension after activation of the TrkA tyrosine kinase receptor by NGF involves complex signaling pathways. Stimulation of SphK1 is part of the functional TrkA signaling repertoire. We found that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation of neurons down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension. In an effort to further understanding of the regulation of SphK1, we used a yeast two-hybrid screen to search for SphK1-interacting proteins that might regulate its activity and/or its cellular localization. One of these was identified as a C-terminal fragment of aminoacylase 1 (Acy1), a metalloenzyme that removes amide-linked acyl groups from amino acids and may play a role in regulating responses to oxidative stress. Acy1 co-immunoprecipitated with SphK1. Though both C-terminal and full-length Acy1 reduced SphK1 activity measured in vitro, the C-terminal fragment inhibited while full-length Acy1 potentiated the growth promoting and anti-apoptotic effects of SphK1. Interestingly, Acy1 expression induced redistribution of SphK1 determined by immunocytochemistry and subcellular fractionation. Our results suggest that Acy1 physically interacts with SphK1 and may influence its physiological functions. Because of the pivotal role of S1P in cells, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by SphKs and degradation by S1P lyase and a specific S1P phosphatase (SPP-1). Surprisingly, we found that down-regulation of SPP-1 enhanced migration of cells towards EGF and conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration towards EGF. We examined whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase. Although the ceramide synthase inhibitor fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. We then examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Collectively, our results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration. We previously found that S1P and ceramide also play important roles in regulating tetrahydrobiopterin synthesis as well as survival of neurons and glial cells. In collaborative studies with Dr. Cory Harding, we examined the role of tetrahydrobiopterin in the efficacy of heterologous muscle-directed gene therapy of PKU with phenylalanine hydroxylase. It appears that the effectiveness of this type of therapy will be limited by the ability to supply sufficient tetrahydrobiopterin to tissues. We also found in collaboration with Dr. R.J. Groszmann that bacterial translocation leads to endotoxemia, stimulation of GTP cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, and enhancement of nitric oxide production which aggravates vasodilation. These results suggest that targeting tetrahydrobiopterin biosynthesis might be beneficial in treatment of septic shock.